CN106520686A - Culture method of adipose tissue-derived stromal cells - Google Patents
Culture method of adipose tissue-derived stromal cells Download PDFInfo
- Publication number
- CN106520686A CN106520686A CN201610887111.8A CN201610887111A CN106520686A CN 106520686 A CN106520686 A CN 106520686A CN 201610887111 A CN201610887111 A CN 201610887111A CN 106520686 A CN106520686 A CN 106520686A
- Authority
- CN
- China
- Prior art keywords
- stem cell
- ctg
- cell
- aac
- factor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2303—Interleukin-3 (IL-3)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/998—Proteins not provided for elsewhere
Abstract
The invention provides a culture method of adipose tissue-derived stromal cells. The culture method of the adipose tissue-derived stromal cells comprises the following culture step: inoculating acquired primary-generation adipose tissue-derived stromal cells into a stem cell culture medium, wherein the stem cell culture medium contains stem cell factors and interleukin-3 factors, and the content ratio of the stem cell factors to the interleukin-3 factors is (5 to 20 [mu]mol/L) to (5 to 20 [mu]mol/L). The culture method can improve the multiplication ability of the adipose tissue-derived stromal cells and achieve relatively high transfer ability.
Description
Technical field
The invention belongs to biological technical field, and in particular to a kind of cultural method of fat mesenchymal stem cell.
Background technology
Mescenchymal stem cell (Mesenchymal Stem Cells, MSCs) is found in marrow earliest, has been proved to deposit
Be body Various Tissues organ in, be multipotential stem cell that a group has multi-lineage potential.With other source MSCs phases
Than adipose-derived mescenchymal stem cell (adipose derived stromal cells, ADSCs) has very on being separately cultured
Big advantage：
(1) draw materials conveniently.Adipose tissue deposit enriches, and pain of drawing materials is little.A current lipsuction can obtain 200ml fat
Fat, isolates about 1 × 106Individual stem cell, is 40 times of marrow fractional dose；(2) separate simple.Periphery fat Jing mechanical shearings,
Collagenase digesting and simple gradient centrifugation are achieved with ADSCs.
But during current practice, the culture of fat mesenchymal stem cell has that amplification ability is limited, animal migration
The problems such as difference.The animal migration of stem cell has been found to closely related with the differentiation capability of stem cell and injury repair ability, therefore,
It is to be highly desirable to that a kind of new fat mesenchymal stem cell cultural method is found to promote its propagation, improve its transfer ability
's.
The content of the invention
A kind of above-mentioned deficiency for aiming to overcome that prior art of the embodiment of the present invention, there is provided fat mesenchymal stem cell
Cultural method, there is the technologies such as amplification ability is limited, animal migration is poor with the culture for solving existing fat mesenchymal stem cell and ask
Topic.
In order to realize foregoing invention purpose, as an aspect of of the present present invention, there is provided a kind of fat mesenchymal stem cell
Cultural method.The primary fat mesenchymal stem cell for obtaining is seeded to dry thin by the cultural method of the fat mesenchymal stem cell
Culture process is carried out in born of the same parents' culture medium, wherein, contains stem cell factor and the interleukin-13 factor in the stem cell media, and
The stem cell factor is (5-20 μm of ol/L) with the content ratio of the interleukin-13 factor:(5-20ug/ml).
Compared with prior art, the cultural method of fat mesenchymal stem cell of the present invention is by adding in stem cell media
Plus stem cell factor and the interleukin-13 factor, and control two factor content ratios so that two cell factors produce dry to fat mesenchymal
The synergistic effect of cell growth and propagation, so as to improve amplification ability of the fat mesenchymal stem cell in incubation, and obtains
Higher transfer ability, the amplification ability for efficiently solving the presence of conventional fat mescenchymal stem cell cultural method is limited, move
The problem of shifting property difference, carries out tissue damage reparation for clinical practice fat mesenchymal stem cell and provides excellent seed.
On the basis of stem cell factor and interleukin-13 factor synergistic effect, by containing for setting up in cultural method
The coating of fibronectin coating buffer is processed so that further produced between fibronectin and stem cell factor and the interleukin-13 factor
Synergistic effect, so that improve propagation and transfer ability of the fat mesenchymal stem cell in incubation.
Description of the drawings
Below in conjunction with drawings and Examples, the invention will be further described, in accompanying drawing：
Fig. 1 is that agarose gel electrophoresis figure is identified in 1 recombinant plasmid pET-30a-rhIL-3 digestions of embodiment；Wherein, M:DNA
ladder；1～3：Plasmid pET-30a-rhIL-3 carries out digestion with Nde I and Xho I；
Fig. 2 is the SDS- of rhIL-3 abduction delivering results in bacterial strain BL21 (DE3) pET-30a-rhIL-3 in embodiment 1
PAGE detection figures；Wherein, 1：Without BL21 (DE3) the pET-30a-rhIL-3 bacterial proteins that IPTG is induced；2：IPTG is induced
1#BL21 (DE3) pET-30a-rhIL-3 bacterial proteins afterwards；3：2#BL21 (DE3) pET-30a- after IPTG inductions
RhIL-3 bacterial proteins；4：The commercialization IL-3 samples of purchase；M：Protein Marker；
SDS-PAGE detection figures of the Fig. 3 for the purpose IL-3 recombinant protein after 1 intermediate ion displacement chromatography of embodiment；Wherein,
M：Albumen Marker；1：BL21 (DE3) pET-30a-rhIL-3 bacterial proteins after IPTG inductions；2：300mmol NaCl are washed
De- thing 1# pipes；3：300mmol NaCl eluates 2# is managed；
Fig. 4 is the ADSC growth curve charts of embodiment 1-2 and each embodiment cultures of comparative example 1-3；
Fig. 5 is the ADSC phenotype flow cytometer detection figures that embodiment 1 and comparative example 3 cultivate；Wherein Fig. 5 A are dry for mesenchyma
Cell surface antigen expresses streaming figure, and Fig. 5 B are hematopoietic cell marker expression streaming figure；
Fig. 6 is the flow cytometer detection figure in the ADSC cycles that embodiment 1 and comparative example 3 cultivate；Wherein Fig. 6 A are Ctr-
The flow cytometer detection figure of ADSC cell cycles；Flow cytometer detection figures of Fig. 6 B for the F/S/I-ADSC cell cycles；Fig. 6 C are Ctr-ADSC
Cell and F/S/I-ADSC cell cycle flow cytometer detection result statistical charts；
Fig. 7 is the ADSC animal migrations detection figure that embodiment 1 and comparative example 3 cultivate；Wherein Fig. 7 A are that Ctr-ADSC is thin
Born of the same parents' traveling locus tracing figure；Fig. 7 B are F/S/I-ADSC cell migration track following figures；Fig. 7 C are Ctr-ADSC cells and F/S/
I-ADSC cell migration average speed statistical charts.
Specific embodiment
In order that the objects, technical solutions and advantages of the present invention become more apparent, it is below in conjunction with drawings and Examples, right
The present invention is further elaborated.It should be appreciated that specific embodiment described herein is only to explain the present invention, and
It is not used in the restriction present invention.
Relevant speciality vocabulary explanation：
Fibronectin (Fibronectin, FN)：The glycoprotein extracellular matrix of HMW (～440kDa), and cell
The receptor protein for being referred to as integrin in film is combined.
Stem cell factor (Stem Cell Factor, SCF)：The part of tyrosine kinase receptor c-Kit.
Interleukin-13 (Interleukin 3, IL-3)：A kind of polyphenic cell factor, mainly by the T cell that have activated,
Macrophage and bone marrow stroma stem cell are produced, and by being combined with its acceptor IL-3R, induce swashing for JAK-STAT5 signal paths
It is living, control DNA synthesis, cell cycle regulation process.
IPTG：Isopropylthiogalactoside, is a kind of extremely strong derivant of effect.
On the one hand, embodiments provide it is a kind of can effectively improve fat mesenchymal stem cell propagation and migrate
Fat mesenchymal stem cell cultural method.The present embodiment fat mesenchymal stem cell cultural method is containing having the following steps：
The primary fat mesenchymal stem cell for obtaining is seeded in stem cell media carries out culture process.
In step, contain stem cell factor and the interleukin-13 factor in the stem cell media, and control described dry thin
Intracellular cytokine is (5-20 μm of ol/L) with the content ratio of the interleukin-13 factor:(5-20ug/ml).It is dry thin by adding in the medium
Intracellular cytokine and the interleukin-13 factor, and control the content ratio of two factors so that amplification of two factors in fat mesenchymal stem cell
Middle performance synergistic effect, so as to improve amplification ability of the fat mesenchymal stem cell in incubation, and obtains higher moving
Shifting ability.
In one embodiment, concentration of the stem cell factor in the stem cell media is controlled for 5-20 μm of ol/ml.When
So under the premise of stem cell factor and interleukin-13 factor content ratio are clear and definite, specify that stem cell factor in the stem cell
Concentration in culture medium, also just specify that concentration of the interleukin-13 in the stem cell media.By optimal control stem cell
The concentration of the factor and interleukin-13 in the stem cell media, can further improve stem cell factor and the interleukin-13 factor
Synergistic effect, so as to further improve amplification ability and the transfer ability of fat mesenchymal stem cell.
On the basis of above-described embodiment, in one embodiment, the interleukin-13 factor is recombined human IL-3, and specifically Jing is excellent
People's total length IL-3 gene after change induced for target gene after recombined human IL-3.Wherein, people's total length IL-3 gene order is
People's total length IL-3 gene order of GenBank data-base recordings, the people's total length IL-3 gene order after the optimization is to be directed to
The optimization that people's total length IL-3 gene order of GenBank data-base recordings is carried out.In a particular embodiment, the people after the optimization
Total length IL-3 gene order is as follows：
5'-ATG AGC CGC CTG CCC GTC CTG CTC CTG CTC CAA CTC CTG GTC CGC CCC
GGA CTC CAA GCT CCC ATG ACC CAG ACA ACG CCC TTG AAG ACA AGC TGG GTT AAC TGC
TCT AAC ATG ATC GAT GAA ATT ATA ACA CAC TTA AAG CAG CCA CCT TTG CCT TTG CTG
GAC TTC AAC AAC CTC AAT GGG GAA GAC CAA GAC ATT CTG ATG GAA AAT AAC CTT CGA
AGG CCA AAC CTG GAG GCA TTC AAC AGG GCT GTC AAG AGT TTA CAG AAC GCA TCA GCA
ATT GAG AGC ATT CTT AAA AAT CTC CTG CCA TGT CTG CCC CTG GCC ACG GCC GCA CCC
ACG CGA CAT CCA ATC CAT ATC AAG GAC GGT GAC TGG AAT GAA TTC CGG AGG AAA CTG
ACG TTC TAT CTG AAA ACC CTT GAG AAT GCG CAG GCT CAA CAG ACG ACT TTG AGC CTC
GCG ATC TTT TGA-3'。——SEQ ID NO 1
Wherein, the induction preparation method of above-mentioned recombined human IL-3 is with the people's total length IL-3 gene order after optimization as target base
Because being designed primer and corresponding restriction enzyme site, then according to the method for routine is converted and is expressed, specifically will optimization
People's total length IL-3 gene order afterwards is inserted in prokaryotic expression carrier, obtains recombinant plasmid, then after converting acquisition containing optimization
People's total length IL-3 gene order bacterial strain, finally realize the expression of recombined human IL-3.In a particular embodiment, recombined human IL-3
Preparation method can with but not only the preparation method of recombined human IL-3 in following article embodiment 1 is prepared.Using above-mentioned
Recombined human IL-3 so that the synergistic effect which is produced between stem cell factor is higher, so that fat mesenchymal stem cell
Amplification ability and transfer ability it is higher.
On the basis of the various embodiments described above, in a preferred embodiment, above-mentioned culture process is the training in coated process
Carry out in foster device, the coating buffer that the coating is processed contains fibronectin.By adopting the coating buffer containing fibronectin in advance
Coating process is carried out to incubator so that function factor fibronectin is distributed in the inside of incubator, when by between primary fat
During mesenchymal stem cells carry out culture process in being seeded to stem cell media, the fibronectin can play a role, specifically
It is their ability in the synergistic effect for participate in stem cell factor and the interleukin-13 factor, so that three factors produce synergistic effect,
So as to improve propagation and the transfer ability of fat mesenchymal stem cell.On this basis, in an embodiment, the fibronectin
Concentration in the coating buffer is 5-20 μ g/ml.In a particular embodiment, coating process method can with but not just for will
During the cushioning liquid of the fibronectin containing the concentration adds incubator, in preferably 4 DEG C lucifuges overnight.
In addition, the acquisition methods of the primary fat mesenchymal stem cell in the various embodiments described above can be according to existing method
Obtain.Primary fat mesenchymal stem cell cultivates the condition of process in stem cell media, and such as cultivation temperature can be using normal
The cultivation temperature of rule, such as 37 DEG C.
In order to effectively play stem cell factor and the interleukin-13 factor or fibronectin, stem cell factor and interleukin-13 because
Effect between son, in one embodiment, in the culture processing procedure in the various embodiments described above, more renews after 24 hours
The stem cell media, changes once the stem cell media afterwards every three days.
On the other hand, above on the basis of the cultural method of embodiment of the present invention fat mesenchymal stem cell, this enforcement
Example additionally provides a kind of fat mesenchymal stem cell, and specifically, the fat mesenchymal stem cell is by the embodiment of the present invention above
The cultural method culture of fat mesenchymal stem cell is obtained.Therefore, embodiment of the present invention fat mesenchymal stem cell has high
Amplification ability and transfer ability.
The present invention is described in further details below by specific embodiment.
Embodiment 1
A kind of cultural method of fat mesenchymal stem cell is present embodiments provided, is comprised the steps：
1. the preparation of recombined human IL-3 (rhIL-3)
The structure of 1.1 pET-30a-rhIL-3 prokaryotic expression bacterial strains
The optimization of base codon is carried out to the people's total length IL-3 gene from GenBank databases so as to be easy to big
Express in enterobacteria.Base sequence after optimization is as follows：
5'-ATG AGC CGC CTG CCC GTC CTG CTC CTG CTC CAA CTC CTG GTC CGC CCC
GGA CTC CAA GCT CCC ATG ACC CAG ACA ACG CCC TTG AAG ACA AGC TGG GTT AAC TGC
TCT AAC ATG ATC GAT GAA ATT ATA ACA CAC TTA AAG CAG CCA CCT TTG CCT TTG CTG
GAC TTC AAC AAC CTC AAT GGG GAA GAC CAA GAC ATT CTG ATG GAA AAT AAC CTT CGA
AGG CCA AAC CTG GAG GCA TTC AAC AGG GCT GTC AAG AGT TTA CAG AAC GCA TCA GCA
ATT GAG AGC ATT CTT AAA AAT CTC CTG CCA TGT CTG CCC CTG GCC ACG GCC GCA CCC
ACG CGA CAT CCA ATC CAT ATC AAG GAC GGT GAC TGG AAT GAA TTC CGG AGG AAA CTG
ACG TTC TAT CTG AAA ACC CTT GAG AAT GCG CAG GCT CAA CAG ACG ACT TTG AGC CTC
GCG ATC TTT TGA-3'——SEQ ID NO 1
RhIL-3 full-length gene orders after optimization are synthesized, (wherein 5 ' hold digestions for design primer and restriction enzyme site
Site is Nde I, and 3 ' end restriction enzyme sites are Xho I), the sequence of forward primer and reverse primer is as follows：
5'-CGCCATATGATGAGCCGCCTGCCCG-3'——SEQ ID NO 2
5'-CTCGAGCGGTCAAAAGATCGCGAG-3'——SEQ ID NO 3
.RhIL-3 full-length genes are inserted in prokaryotic expression carrier pET-30a (+) carrier, recombinant plasmid pET- is obtained
30a-rhIL-3, and converted in BL21 (DE3).On LB plates after kanamycins (30 μ g/ml) resistance screening,
Picking single bacterium colony is cultivated in LB culture mediums, and extracting plasmid carries out digestion identification, and the product Jing agaroses after digestion are coagulated
Gel electrophoresis identify that qualification result is as shown in figure 1, it follows that artificial synthesized rhIL-3 full length sequences (459bp) are successfully inserted
In entering prokaryotic expression carrier pET-30a (+) (5422bp), and convert in BL21 (DE3), that is, the positive expression bacterial strain for obtaining
BL21(DE3)pET-30a-rhIL-3。
The abduction delivering of 1.2 IL-3 recombinant proteins
According to 1:50 ratio is inoculated with BL21 (DE3) pET-30a-rhIL-3 bacterial classifications to LB culture mediums (containing 30ug/ml
Kan in), 37 DEG C, 220-250rpm concussion and cultivates overnight.The next morning is according to 1:100 ratio is inoculated with the previous day culture
Bacterium Amplification Culture in LB nutrient solutions (Kan containing 30ug/ml), to OD600=0.5-0.6 (general inoculated and cultured 2-3h),
IPTG to final concentration of 1mM, 30 DEG C of abduction delivering 3h are added, thalline is harvested, destination protein is detected with SDS-PAGE, reflected
Determine result as shown in Figure 2.As shown in Figure 2, the result of swimming lane 2,3 shows, bacterial strain BL21 (DE3) pET-30a-rhIL-3 are at 30 DEG C
Being capable of successful expression rhIL-3 when being induced with 1mM IPTG.
The purifying of 1.3 IL-3 recombinant proteins
By the bacteria suspension 5000g of above-mentioned collection, 4 DEG C of centrifugation 10min, supernatant is abandoned, with the lysis buffer of precooling
40-50 times of (50mMPB, 5mMEDTA, pH6.5) concentrates thalline, is placed in 50ml centrifuge tubes or 50ml beakers, carries out ultrasound broken
It is broken.Ultrasound condition：Ice-water bath, ultrasonic 4s are spaced 5s, 4min/ time, repeat to bacterium solution change clarification.By lysate 12000g,
4 DEG C centrifugation 10min, collect supernatant, successively with 0.8um, 0.45 μm of membrane filtration after, carried out using ion exchange chromatography
Sample is purified and is concentrated, and rough flow is as follows：
(1) pillar being washed with 20mM PB, being carried out with wash-out, 280nm absorbances are first raised and reduced afterwards, are down to 0.000-
Change an eluent to be eluted when 0.005；
(2) the 20mM PB of 180mM NaCl wash pillar, and 280nm absorbances are first raised and reduced afterwards, are down to 0.000-
Use next eluent when 0.007 instead；
(3) the 20mM PB containing 300mM NaCl elute destination protein, are in charge of collection.Simultaneously with SDS-PAGE to collection
Destination protein is detected；The electrophoretogram of detection is shown in Fig. 3.From Fig. 31,2,3 swimming lanes can be seen that and can be obtained using the method
Obtain highly purified rhIL-3 albumen；
(4) the destination protein sample for taking 50ml collections adds PB buffer solutions of the 100ml without NaCl, enters in 4 DEG C of refrigerators
Row ultrafiltration, stops ultrafiltration as liquid residual 20ml or so in ultrafiltration cup, opens ultrafiltration cup, suction out a small amount of sample in super-clean bench
Solution, detects protein concentration, by sample concentration to 1mg/ml, is filtered with the filter of 0.22um, and is dispensed standby.
2. primary fat mesenchymal stem cell is separately cultured
2.1 Day 0：
The coating of Tissue Culture Flask：1 T75 blake bottle is taken, DPBS solution of the 5ml containing 10ug/ml FN, 4 DEG C of lucifuges are added
Overnight place.
The preparation of 1# complete mediums：DMEM in high glucose+2%Ultroser G+10 μm ol/L SCF+10ug/ml IL-3+
1% mycillin.Wherein, IL-3 is recombined human IL-3 (rh IL-3) that obtains in step 1.
2.2 Day 1：225ml centrifuge tubes are injected after operating room liposuction, and centrifuge tube is placed in ice chest, reality is transported to
Test room.
2.3 rinse fat with the DPBS containing 0.1% gentamicin removes anesthetic and blood.
2.4 by upper-layer fat, by 10ml, often pipe is transferred to 50ml centrifuge tubes, and often pipe adds 0.1% type i collagen enzyme 20ml,
It is placed in 37 DEG C of water bath with thermostatic control shaking tables, 200rpm concussion 60min, period concussion are mixed once, taken out, three layers of liquid level point, upper strata
For oil layer, middle for not digesting fat tissue layer, lower floor is fat mesenchymal cell and red blood cell mixed layer.
2.5 add 20ml to terminate culture medium (DMEM in high glucose+10%FBS+1% mycillins), 1500rpm centrifugation 15min.
2.6 that upper two-layer is transferred to new centrifuge tube is standby, abandons lower floor's mixed liquor, remains cell precipitation, adds 20ml DPBS
Re-suspended cell.If (upper two layers of fat particle is more, can add 0.1% type i collagen enzyme 20ml again, repeats aforesaid operations).
2.7 it is resuspended after cell suspension with 100um cell screen clothes filter, 1500rpm centrifugation 15min after, abandon supernatant, use 1#
After complete medium is resuspended, proceed in the T75 blake bottles for discarding coating buffer, be placed in 37 DEG C, 5%CO2Cultivate in incubator.
A large amount of cell attachments are observed after 2.8 24h, are changed fresh complete medium, are changed liquid once per two days afterwards,
When growth is fused to 90%, with 0.25% pancreatin had digestive transfer culture culture.The cell marking that the culture of 1# complete mediums is obtained is F/
S/I-ADSC。
Embodiment 2
A kind of cultural method of fat mesenchymal stem cell is present embodiments provided, is comprised the steps：
1. the preparation of recombined human IL-3 (rhIL-3), with reference to the step of embodiment 11；
2. primary fat mesenchymal stem cell is separately cultured
2.1 Day 0：
Tissue Culture Flask：1 T75 blake bottle is taken, coating process is not carried out.
The preparation of 1# complete mediums：DMEM in high glucose+2%Ultroser G+10 μm ol/L SCF+10ug/ml IL-3+
1% mycillin.Wherein, IL-3 is recombined human IL-3 (rh IL-3) that obtains in step 1.
Step 2.2 to step 2.8 with reference to the step of embodiment 1 2.2 to step 2.8；The cell marking that culture is obtained is S/
I-ADSC。
Comparative example 1
A kind of cultural method of fat mesenchymal stem cell is present embodiments provided, is comprised the steps：
1. the preparation of recombined human IL-3 (rhIL-3)：With reference to the step 1 in embodiment 1.
2. primary fat mesenchymal stem cell is separately cultured
2.1 Day 0：
Tissue Culture Flask：1 T75 blake bottle is taken, coating process is not carried out.
The preparation of 2# complete mediums：DMEM in high glucose+2%Ultroser G+10ug/ml IL-3+1% mycillins.Its
In, IL-3 is recombined human IL-3 (rh IL-3) that obtains in step 1.
Step 2.2 to step 2.6 with reference to the step of embodiment 1 2.2 to step 2.6；
2.7 it is resuspended after cell suspension with 100um cell screen clothes filter, 1500rpm centrifugation 15min after, abandon supernatant, use 2#
After complete medium is resuspended, proceed in T75 blake bottles, be placed in 37 DEG C, 5%CO2Cultivate in incubator.
A large amount of cell attachments are observed after 2.8 24h, are changed fresh complete medium, are changed liquid once per two days afterwards,
When growth is fused to 90%, with 0.25% pancreatin had digestive transfer culture culture.The cell marking that the culture of 2# complete mediums is obtained is I-
ADSC。
Comparative example 2
A kind of cultural method of fat mesenchymal stem cell is present embodiments provided, is comprised the steps：
1. primary fat mesenchymal stem cell is separately cultured
1.1 Day 0：
Tissue Culture Flask：1 T75 blake bottle is taken, coating process is not carried out.
The preparation of 3# complete mediums：DMEM in high glucose+2%Ultroser G+10 μm ol/L SCF+1% mycillins.
Step 1.2 to step 1.6 with reference to the step of embodiment 1 2.2 to step 2.6；
1.7 it is resuspended after cell suspension with 100um cell screen clothes filter, 1500rpm centrifugation 15min after, abandon supernatant, use 3#
After complete medium is resuspended, proceed in T75 blake bottles, be placed in 37 DEG C, 5%CO2Cultivate in incubator.
A large amount of cell attachments are observed after 1.8 24h, are changed fresh complete medium, are changed liquid once per two days afterwards,
When growth is fused to 90%, with 0.25% pancreatin had digestive transfer culture culture.The cell marking that the culture of 3# complete mediums is obtained is S-
ADSC。
Comparative example 3
A kind of cultural method of fat mesenchymal stem cell is present embodiments provided, is comprised the steps：
1. primary fat mesenchymal stem cell is separately cultured
1.1 Day 0：
Tissue Culture Flask：1 T75 blake bottle is taken, coating process is not carried out.
The preparation of 4# complete mediums：DMEM in high glucose+2%Ultroser G+1% mycillins.
Step 1.2 to step 1.6 with reference to the step of embodiment 1 2.2 to step 2.6；
1.7 it is resuspended after cell suspension with 100um cell screen clothes filter, 1500rpm centrifugation 15min after, abandon supernatant, use 4#
After complete medium is resuspended, proceed in T75 blake bottles, be placed in 37 DEG C, 5%CO2Cultivate in incubator.
A large amount of cell attachments are observed after 1.8 24h, are changed fresh complete medium, are changed liquid once per two days afterwards,
When growth is fused to 90%, with 0.25% pancreatin had digestive transfer culture culture.The culture of 4# complete mediums obtain cell marking be
Ctr-ADSC。
Related experiment is tested and test result
The I- that F/S/I-ADSC, S/I-ADSC and comparative example 1-3 that above-described embodiment 1-2 is obtained is obtained respectively
ADSC, S-ADSC, Ctr-ADSC carry out following related experiments respectively：
1. fat mesenchymal stem cell growth curve is drawn
In taking each embodiment respectively, P4 is for rear each ADSC cells, with 1 × 104The density in/hole is inoculated with 24 orifice plates, every two
My god, holes cell is taken, and cell suspension is collected after pancreatin digestion, is carried out cell count with cell counting count board, take holes cell number equal
Value, draws the cell growth curve of different groups.Jing tests learn that the multiplication capacity of the ADSC cells that embodiment 1-2 is obtained is better than
The multiplication capacity of the Ctr-ADSC cells that the ADSC cultivated in comparative example 1-3, wherein comparative example 3 are obtained is most weak,
The growth curve of concrete ADSC is as shown in Figure 4.Thus demonstrate between SCF and recombined human IL-3, SCF and recombined human IL-3 and FN
Synergistic effect with the propagation to ADSC and growth between three.
2. fat mesenchymal stem cell phenotypic evaluation
In each embodiment of 2.1 digestion, P4 is counted for the ADSC cells of rear exponential phase；
2.2 take 10 respectively6In streaming pipe, DPBS is washed twice individual cell, and supernatant is removed in centrifugation；
After 2.3 100ul DPBS are resuspended, CD90-FITC, CD105-FITC, CD45-PE, CD34-PE is added separately to
Antibody, normal temperature lucifuge incubation 30min；
2.4 DPBS are washed twice, and supernatant is removed in centrifugation；
2.5 0.5ml DPBS re-suspended cells, flow cytomery cell phenotype, Cell-Quest software analysis, each
Sample at least analyzes 10000 cells.
The ADSC cells of P4 exponential phases for after in taking each embodiment respectively, with flow cytomery cell table
The expression of face CD90, CD105, CD45, CD34 antigen, flow cytometer detection result show that ADSC cell surfaces are low in each embodiment
Expression hematopoietic cell mark CD34, CD45, high expression mescenchymal stem cell surface antigen CD90, CD105.Wherein, with enforcement
As a example by the Ctr-ADSC cells of the F/S/I-ADSC cells of example 1 and comparative example 3, its respective flow cytometer detection result such as Fig. 5 institute
Show, the equal low expression hematopoietic cell mark CD34 of two groups of ADSC cell surfaces, CD45 (<2%), high expression mescenchymal stem cell table
Face antigens c D90, CD105 (>95%).
3. ADSCs of the P4 for rear exponential phase in the 3.1 each embodiment of digestion of fat mesenchymal stem cell cell cycle detection
Cell, counts；
3.2 take 10 respectively6In streaming pipe, DPBS is washed twice individual cell, and supernatant is removed in centrifugation；
3.3 shake while adding the mixing of 70% pre-cooled ethanol, 4 DEG C of fixed more than 18h in cell precipitation；
3.4 centrifugations, discard ethanol supernatant, add the DPBS of precooling to wash and precipitate 2 times, and supernatant is removed in centrifugation；
3.5 0.5ml DPBS re-suspended cells, add the 5mg/ml RNases (final concentration 50ug/ml) of 5ul, incubate at 37 DEG C
Educate 30min；
3.6 add 25ul 1mg/ml PI (final concentration 50ug/ml), room temperature, lucifuge reaction 30min after mixing；
3.7 flow cytometers carry out DNA detections, with the ratio of Modifit software analysis each phase cell cycle.
In taking each embodiment respectively, the ADSC cells of P4 exponential phases for after, carry out DNA inspections with flow cytometer
Survey, with the ratio of Modifit software analysis each phase cell cycle, experimental result shows the embodiment 1-2 training in the G2-M phases
Foster F/S/I-ADSC, S/I-ADSC cell ratio is significantly larger than I-ADSC, S-ADSC, Ctr- in comparative example 1-3
ADSC, and difference has conspicuousness (P<0.05), further demonstrate that between SCF and recombined human IL-3, SCF and recombined human IL-3 and
Synergistic effect with the propagation to ADSC and growth between FN three, through SCF and two factors of IL-3 or SCF, IL-3 and FN
The multiplication capacity of the ADSC cells of three factor treatments is better than the Ctr-ADSC cells cultivated by conventional method.With embodiment 1
F/S/I-ADSC cell proportions as a example by the Ctr-ADSC of F/S/I-ADSC and comparative example 3, in embodiment 1 in the G2-M phases
For 19.8%, the significantly larger than cell proportion (6.7%) of the Ctr-ADSC cell G2-M phases of comparative example 3, and both differences
It is different to have conspicuousness (P<0.05), as shown in Figure 6.
4. fat mesenchymal stem cell transfer ability detection
4.1 take P4 respectively for rear Ctr-ADSC cells and F/S/I-ADSC cells, with 1x105/ hole is seeded to confocal
Special chamber, is placed in 37 DEG C, 5%CO23h is cultivated in incubator so that cell attachment；
4.2 take out chamber, using 1ml syringe needles, rule in bottom surface central authorities, change carbon dioxide it is non-according to
The culture medium of bad property, experimental group cell add 10ug/ml IL-3, control group to add equivalent DPBS；
4.3 are placed in living cells work station chamber to carrying out being continuously shot observation at line, arrange under 10 times of object lens
A photo was shot per two minutes, was continuously shot 24h；
Photo captured by 4.4 selects one every ten, is processed as video using ImageJ software processings；
4.5 in ImageJ to line at one end randomly select 20 cell movement tracks and be tracked, count two groups thin
Born of the same parents' movement rate difference.
The ADSC cell movement speed differences after P4 generations in counting each embodiment respectively, test result indicate that, embodiment
1-2 culture F/S/I-ADSC, S/I-ADSC cell migration be better than the I-ADSC in comparative example 1-3, S-ADSC,
Ctr-ADSC, and difference has conspicuousness (P<0.05), further demonstrate that between SCF and recombined human IL-3, SCF and recombined human
There is the synergistic effect for promoting ADSC transfer abilities, through SCF and two factors of IL-3 or SCF, IL-3 between IL-3 and FN threes
It is better than the Ctr-ADSC cells cultivated by conventional method with the transfer ability of the ADSC cells of tri- factor treatments of FN.With embodiment
As a example by the 1 F/S/I-ADSC and Ctr-ADSC of comparative example 3, the F/S/I-ADSC transfer abilities of embodiment 1 are real with contrast
The Ctr-ADSC for applying example 3 is compared, and both migration velocities have significant difference (P<0.05), as shown in Figure 7.
Presently preferred embodiments of the present invention is the foregoing is only, not to limit the present invention, all essences in the present invention
Any modification, equivalent and improvement made within god and principle etc., all should be included within protection scope of the present invention.
Claims (6)
1. a kind of cultural method of fat mesenchymal stem cell, it is characterised in that including following incubation step：
The primary fat mesenchymal stem cell for obtaining is seeded in stem cell media carries out culture process, wherein, described dry
Contain stem cell factor and the interleukin-13 factor in cell culture medium, and the content ratio of the stem cell factor and the interleukin-13 factor
For (5-20 μm of ol/L):(5-20ug/ml).
2. cultural method according to claim 1, it is characterised in that：The culture process is the culture in coated process
Carry out in device, the coating buffer that the coating is processed contains fibronectin.
3. cultural method according to claim 2, it is characterised in that：Concentration of the fibronectin in the coating buffer
For 5-20 μ g/ml.
4. according to the arbitrary described cultural method of claim 1-3, it is characterised in that：The interleukin-13 factor is with optimized
People's total length IL-3 gene afterwards induced for target gene after recombined human IL-3, the people's total length IL-3 gene sequence after the optimization
Row are as follows：
5'-ATG AGC CGC CTG CCC GTC CTG CTC CTG CTC CAA CTC CTG GTC CGC CCC GGA
CTC CAA GCT CCC ATG ACC CAG ACA ACG CCC TTG AAG ACA AGC TGG GTT AAC TGC TCT
AAC ATG ATC GAT GAA ATT ATA ACA CAC TTA AAG CAG CCA CCT TTG CCT TTG CTG GAC
TTC AAC AAC CTC AAT GGG GAA GAC CAA GAC ATT CTG ATG GAA AAT AAC CTT CGA AGG
CCA AAC CTG GAG GCA TTC AAC AGG GCT GTC AAG AGT TTA CAG AAC GCA TCA GCA ATT
GAG AGC ATT CTT AAA AAT CTC CTG CCA TGT CTG CCC CTG GCC ACG GCC GCA CCC ACG
CGA CAT CCA ATC CAT ATC AAG GAC GGT GAC TGG AAT GAA TTC CGG AGG AAA CTG ACG
TTC TAT CTG AAA ACC CTT GAG AAT GCG CAG GCT CAA CAG ACG ACT TTG AGC CTC GCG
ATC TTT TGA-3'。
5. according to the arbitrary described cultural method of claim 1-3, it is characterised in that：The stem cell factor is in the stem cell
Concentration in culture medium is 5-20 μm of ol/ml.
6. according to the arbitrary described cultural method of claim 1-3, it is characterised in that：In the culture processing procedure, 24 hours
The stem cell media for more renewing afterwards, changes once the stem cell media afterwards every three days.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610887111.8A CN106520686A (en) | 2016-10-11 | 2016-10-11 | Culture method of adipose tissue-derived stromal cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610887111.8A CN106520686A (en) | 2016-10-11 | 2016-10-11 | Culture method of adipose tissue-derived stromal cells |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106520686A true CN106520686A (en) | 2017-03-22 |
Family
ID=58331237
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610887111.8A Pending CN106520686A (en) | 2016-10-11 | 2016-10-11 | Culture method of adipose tissue-derived stromal cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106520686A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107034184A (en) * | 2017-05-04 | 2017-08-11 | 济南赛尔生物科技股份有限公司 | A kind of kit for original cuiture umbilical cord mesenchymal stem cells |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101984048A (en) * | 2010-11-24 | 2011-03-09 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Culture medium for culturing mesenchymal stem cells |
| WO2011106775A3 (en) * | 2010-02-27 | 2011-11-03 | Reza Izadpanah | Derivation of hematopoietic cells from adult mesenchymal stem cells |
| CN104136034A (en) * | 2011-11-30 | 2014-11-05 | 先进细胞技术公司 | Mesenchymal stromal cells and uses related thereto |
| CN104974984A (en) * | 2015-04-13 | 2015-10-14 | 深圳市默赛尔生物医学科技发展有限公司 | Adipose tissue-derived mesenchymal stem cell amplification culture method |
-
2016
- 2016-10-11 CN CN201610887111.8A patent/CN106520686A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011106775A3 (en) * | 2010-02-27 | 2011-11-03 | Reza Izadpanah | Derivation of hematopoietic cells from adult mesenchymal stem cells |
| CN101984048A (en) * | 2010-11-24 | 2011-03-09 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Culture medium for culturing mesenchymal stem cells |
| CN104136034A (en) * | 2011-11-30 | 2014-11-05 | 先进细胞技术公司 | Mesenchymal stromal cells and uses related thereto |
| CN104974984A (en) * | 2015-04-13 | 2015-10-14 | 深圳市默赛尔生物医学科技发展有限公司 | Adipose tissue-derived mesenchymal stem cell amplification culture method |
Non-Patent Citations (2)
| Title |
|---|
| KRISZTINA SZÖKE等: ""Ectopic expression of CDX4 in human mesenchymal stem cells does not affect HOX gene expression or induce hematopoietic reprogramming"", 《STEM CELL RESEARCH》 * |
| NAOMI OGURA等: ""Differentiation of the human mesenchymal stem cells derived from bone marrow and enhancement of cell attachment by fibronectin"", 《JOURNAL OF ORAL SCIENCE》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107034184A (en) * | 2017-05-04 | 2017-08-11 | 济南赛尔生物科技股份有限公司 | A kind of kit for original cuiture umbilical cord mesenchymal stem cells |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US6541249B2 (en) | Immortalized human stromal cell lines | |
| JP6607675B2 (en) | Mesodermal lineage progenitor cells | |
| Xiao Ling et al. | Stromal derived factor-1/CXCR4 axis involved in bone marrow mesenchymal stem cells recruitment to injured liver | |
| Kaiser et al. | BM cells giving rise to MSC in culture have a heterogeneous CD34 and CD45 phenotype | |
| AU5436998A (en) | MSC-megakaryocyte precursor composition and method of isolating MSCs asso ciated with isolated megakaryocytes by isolating megakaryocytes | |
| EP1718735A1 (en) | Stem cell populations and methods of use | |
| CN102115730A (en) | High metastatic potential hepatoma cell line capable of steady autophagy indication, and establishment method and application method thereof | |
| CN106479968A (en) | The cultural method of umbilical cord mesenchymal stem cells | |
| CN103301154B (en) | Application of umbilical cord mesenchymal stem cells in preparation of formulation for treating lupus erythematosus | |
| CN108611322A (en) | Method for building up and the application of breast cancer circulating tumor cell system CTC-3, culture medium and CTC-3 | |
| CN109988746A (en) | A kind of mescenchymal stem cell adipogenic induction differentiation method | |
| CN104480068A (en) | Method of in-vitro amplification and purification culture of mesenchymal stem cells | |
| CN106520686A (en) | Culture method of adipose tissue-derived stromal cells | |
| CN108486039A (en) | The method that small molecule induction human adipose-derived stem cell is divided into interstitial glands | |
| CN105441386A (en) | Culture and identification method for very small porcine embryonic-like stem cells | |
| CN105505858A (en) | Separation and purification method for very small embryonic-like stem cells (VSELs) of pigs | |
| US9045735B2 (en) | Enrichment of tissue-derived adult stem cells based on retained extracellular matrix material | |
| CN104995295A (en) | A method of generating multilineage potential cells | |
| CN106497883A (en) | A kind of human umbilical cord mesenchymal stem cells of stably expression antioncogene REIC/Dkk3 and its construction method and application | |
| CN103266084B (en) | Three-step separation method for enriching stem cells | |
| Ribeiro et al. | Quantification and comprehensive analysis of mesenchymal stromal cells in bone marrow samples from sickle cell disease patients with osteonecrosis | |
| Gordeev et al. | Methods for screening live cells | |
| CN104531621A (en) | Separation method of liver cancer stem cells | |
| JP5350364B2 (en) | Cell culture method by signal control between cells using powdered scaffold | |
| CN109810943A (en) | One boar muscle-derived mescenchymal stem cell isolation medium and isolated culture method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170322 |